Optimization of the harvesting and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy We evaluated the utility of a novel protocol involving flt-3 and G-CSF mobilization of peripheral blood stem cells and retroviral transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24, into hematopoietic stem cells in nonhuman primates Rhesus macaques were treated with flt-3 ligand (200 mg/kg) and G-CSF (20 mg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis CD34+ cells were transduced with an MFG-based retroviral vector encoding murine CD24 using 4 daily transductions with centrifugations in the presence of flt-3 ligand (100 ng/ml), SCF (100 ng/ml) and PIXY (100 ng/ml) in serum free medium Animals were sublethally irradiated (400 cGy) on the day of transplantation Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flo w cytometry and semi-quantitative DNA PCR Our data demonstrate successful and persistent engraftment in multiple hematopoietic lineages, including granulocytes, monocytes and B and T lymphocytes At 46 and 117 days post transplantation 75% and 6% of granulocytes, respectively, expressed mCD24 antigen at the cell surface Peak in vivo levels of genetically-modified peripheral blood lymphocytes (PBL) approached 60% as assessed both by flow cytometry and PCR 18 days post transplantation In addition, naive (CD45RA+ and CD62L+) CD4+ and CD3+8+ cells were the predominant phenotype of the T cells detected at this time Engraftment has subsequently plateaued but persisted at between 10 and 15% of PBLs for 133 days Acytotoxic T cell response against murine CD24 was detected in only one animal This degree of persistent long-lived, high level gene marking of multiple hematopoietic lineages, including naive T cells, has important implications for the field of stem cell gene therapy